Recently, with the significant progress in the technique of utilizing microorganisms or enzymes produced therefrom, numerous processes for immobilizing enzymes or microbial cells have been proposed and many inventions have been made to utilize enzyme reactions effectively. However, only a few of them have succeeded in industrialization. That is, besides the problem of manufacturing cost, many of the techniques hitherto developed involve various disadvantages, e.g., severe conditions are required to immobilize enzymes or microbial cells, that there is a problem that the bioactivities of the enzymes or microbial cells are reduced upon immobilization using polyfunctional reagents; there is a problem in the physical tolerance of immobilized enzymes or microbial cells whose low mechanical strength cannot tolerate industrial use and may cause clogging of pores due to the deformation by liquid pressure of the substrate; and there is a problem in the safety of immobilized enzymes or microbial cells, or their products, which are obtained by using harmful monomers, cross linking agents or the like for the immobilization.
Hitherto, various processes for immobilizing enzymes or microbial cells by means of PVA are known. For instance:
(1) A process for immobilizing enzymes by gelating at low temperatures an aqueous solution containing PVA and an enzyme has been proposed (Japanese Patent Application (OPI) No. 52276/75). (The term "OPI" as used herein means a "published unexamined Japanese Patent application".) The gel obtained by this process, however, does not show any elasticity and its mechanical strength is very low. Moreover, only a weak or soft gel is obtained when air-drying is effected after the solidification and fusion, and only a fragile gel hardly showing any elasticity is obtained even when dehydration under reduced pressure is carried out or dehydration is effected over a long period of time, instead of the air-drying. Therefore, this process is not an industrially valuable process.
(2) A process for immobilizing enzymes or microbial cells by instantly gelating an aqeous solution of enzymes or microbial cells by adding thereto an aqueous solution of boric acid or borax has been proposed (Japanese Patent Publication No. 51552/80, Japanese Patent Application (OPI) No. 135293/79). The gel obtained by this process, however, is weak and hardly molded.
(3) A process of immobilization by adding an acid to a suspension containing PVA, tetraethyl silicate and microbial cells and air-drying the mixture has also been proposed (U.S. Pat. No. 4,148,689). The membrane obtained, however, is also weak. Even when freezing and drying are effected after the addition of acid, the membrane produced is reduced in its mechanical strength and is almost incapable of being molded.
(4) A process for immobilizing living microbial cells in a gel by drying an aqueous suspension containing an aqueous PVA solution, living microbial cells and a clay mineral at a temperature of from -6.degree. to +40.degree. C. to dehydrate it until a gel of prescribed moisture content is formed, has also been proposed (Japanese Patent Application (OPI) No. 138390/82). The immobilized microbial cells obtained by this process, however, are still weak and fragile, and does not reach an industrially available strength. In addition, they have a defect in that their activity per volume is lowered because of the clay mineral is employed in a large amount and accordingly the volume of a reactor for the enzyme reaction increases significantly.
(5) A process for immobilizing microbial cells in a gel by freezing and molding an aqueous suspension containing an aqueous PVA solution, living microbial cells and a clay mineral, and then vacuum-drying the molded frozen suspension without melting it, i.e., by so-called freeze-drying, has been proposed (No. EP-60052-A and Canadian Pat. No. 1180670). Likewise, a process for immobilizing microbial cells in a gel by freezing and molding an aqueous suspension containing an aqueous solution and living microbial cells and thereafter immobilizing the cells in the gel formed without causing melting, has been proposed (No. EP-60052-A and Canadian Pat. No. 1180670). The immobilized microbial cells obtained by these processes are relatively firm and reveal a high bioactivity. However, they are disadvantageous in that, since they are obtainable only in a voluminous form and their volume increases further as a large amount of the clay mineral is incorporated, the activity of micobial cells per unit volume decreases and the volume of a reactor increases significantly as they are utilized in enzyme reactions. In these processes, the suspension is frozen and molded in a plate mold having fine pores and then freeze-dried, without being molten, to give a plate molded gel. However, there is a defect in that fine granulation cannot be effected at the stage where the suspension is frozen and molded because the suspension will be defrozen if granulation is tried. Further, there is a defect in that the high cost of the equipment and energy (power for dehydration) is necessary for freeze-drying, as a large amount of moisture is removed by the freeze-drying.
Generally speaking, molding of a mixture of aqueous PVA solution and an enzyme or microbial cells is effected by a process wherein the molding is effected after the PVA gel is formed or by a process wherein the molding is effected by pouring the mixture into a container or mold for molding of any desired form before the PVA gel is formed. In the molding process effected after the formation of PVA gel, it is difficult to obtain a molding of desired form due to the tackiness of the gel formed, and moreover it is impossible to obtain an immobilized enzyme or microbial cell having an industrially sufficient strength (the processes (1) to (4) mentioned above). Also in the molding process effected before the formation of PVA gel wherein a mixture of aqueous PVA solution and living microbial cells is poured into a container or mold for molding of any desired form, frozen and freeze-dried, it is difficult to obtain granular immobilized microbial cells having a diameter of 1 to 3 mm which are used industrially for enzyme reactions, not to speak of the enormous costs of the equipment and energy for the freeze-drying.